Abstract

Spiggin is an adhesive glycoprotein produced in the kidney of sticklebacks during
the breeding season and is subsequently secreted into the urinary bladder from where
it is employed for nest building. Since the production of the protein has been shown
to be under androgenic control, spiggin has been suggested to be a useful biomarker
for androgenic substances in the environment. In this study, two polyclonal spiggin
antibodies based on synthetic peptides and one polyclonal antibody directed against
native spiggin have been characterized. The antibodies ability to identify spiggin
was investigated by quantitative immunoassay. For both peptide antibodies the quantification
range was determined to be between 1 and 80 ng spiggin and determination of renal
spiggin levels from immature and mature males displayed a 15-fold increase in total
spiggin content of the kidney resulting in a 6-fold increase in male kidney weight
due to hypertrophy. The kidney somatic index (KSI) was found to correlate well with
the total renal spiggin content and therefore it appears that KSI in sticklebacks
could be used as an initial method to identify substances displaying androgenic effects.
Furthermore, western blot analysis revealed that the polyclonal antibodies recognize
different spiggin isoforms and that spiggin can be detected in the urinary bladder
and kidney of both males and female sticklebacks. In order to develop a quantitative
detection method for native spiggin it is necessary to produce a standard that can
be used in a bioassay. Due to the adhesive and polymerization characteristics of spiggin
the protein is difficult to use as a standard in bioassays. So far spiggin has been
shown to exist in at least 14 isoforms, all of which contain polymerization domains.
To overcome the solubility problem we have produced recombinant spiggin gamma, with
only one polymerization domain, that can be expressed in E. coli. Western blot analysis
demonstrated that the polyclonal antibodies were able to detect recombinant spiggin
gamma protein in bacterial cell lysate, suggesting that it may be developed into a
useful source of standard spiggin to be used for quantitative determination of androgen
induced spiggin production in sticklebacks.